The invention relates to carbohydrate chemistry and vaccinology.
Parasites of animals and humans pose a worldwide problem. For example, schistosomiasis, after malaria, is the most common cause of human morbidity and mortality. Approximately 600 million people are at risk for schistosome flatworm infection, and approximately 200 million people in 74 countries are infected. Twenty million people (mostly children) have a severe form of the disease, and 200,000 die annually from the disease.
Mammalian parasites, such as platyhelminths of the genus Fasciola or Schistosome and protozoans of the genus Trichomonas, can avoid immune elimination and survive for months or years in the fully immunocompetent vertebrate host. The surface of these parasites elicits a T cell-independent immune response characterized by the predominant production of IgM antibodies but fails to induce a T cell-independent response characterized by the production of IgG, IgE, and IgA isotype antibodies.
The production of IgG (as well as IgE and IgA) and their binding to the exterior of a pathogen is generally required for antibody-dependent cell-mediated cytotoxicity, a mechanism demonstrated to be effective in destroying parasitic worms. The binding of thymus-dependent antibodies (IgG, IgE, and IgA) to the exterior of extracellular pathogens is also generally required for phagocytosis by host macrophages and other immune functions included in a process of immune activation called xe2x80x9copsonizationxe2x80x9d. Opsonization is an immune mechanism frequently associated with destruction of extracellular protozoan parasites. It is generally believed that several mammalian parasites evade immune elimination by failing to induce surface-specific T cell-dependent functions, such as IgG, IgE, and IgA production.
The invention is based on the isolation of new lipoglycans from the surface of platyhelminths. These lipoglycans, as well as those isolated from certain protozoan parasites, can be used in compositions for inhibiting, treating, or diagnosing parasitic infection.
Accordingly, the invention features a method of eliciting in a vertebrate a protective immune response (e.g., one including a T cell-dependent antibody response) against an eukaryotic parasite by administering to the vertebrate a composition containing a carrier group coupled to an oligosaccharide (or a mixture of oligosaccharides) obtained from a lipoglycan found on the surface of an eukaryote. The composition is administered in an amount sufficient to elicit a protective immune response against the eukaryotic parasite.
The oligosaccharide can be isolated or obtained from a lipoglycan (i.e., a molecule having at least one lipid group and at least one carbohydrate group) having a molecular weight of about 180 kilodaltons. In addition, the lipoglycan includes at least one lipid group and at least one carbohydrate group. For example, the lipoglycan can include a lipid group, one or more fucose groups, three to five galactoseamine groups per fucose group, two to four glucosamine groups per fucose group, one to two galactose groups per fucose group, one to two glucose groups per fucose group, one to two rhamnose groups per fucose group, and one to three mannose groups per fucose group. Alternatively, the lipoglycan can include a lipid group, one or more fucose groups, three to five galactoseamine groups per fucose group, seven to eleven glucoseamine groups per fucose group, three to five galactose groups per fucose group, one to two glucose groups per fucose group, and three to five mannose groups per fucose group.
The eukaryote can be a protozoan or an adult platyhelminth (e.g., of the genus Schistosoma or Fasciola, or of the class cestoidea). The eukaryotic parasite can be a protozoan or a pathogenic platyhelminth (e.g., of the genus Schistosoma or Fasciola, or of the class cestoidea).
The carrier group can be coupled to the oligosaccharide by a linker (e.g., 2-(4-amino-phenyl)ethylamine).
The invention also includes an isolated lipoglycan (e.g., one about 180 kDa in size) including a lipid group, one or more fucose groups, three to five galactoseamine groups per fucose group, two to four glucoseamine groups per fucose group, one to two galactose groups per fucose group, one to two glucose groups per fucose group, one to two rhamnose groups per fucose group, and one to three mannose groups per fucose group. In a specific embodiment, this lipoglycan includes, per each fucose group, four galactosamine groups, three glucosamine groups, two galactose groups, two glucose groups, and two mannose groups. The lipoglycan can be obtained from a species of the genus Schistosoma.
The invention further includes an isolated lipoglycan (e.g., one about 180 kDa in size) having a lipid group, one or more fucose groups, three to five galactoseamine groups per fucose group, seven to eleven glucoseamine groups per fucose group, three to five galactose groups per fucose group, one to two glucose groups per fucose group, and three to five mannose groups per fucose group. In a specific embodiment, the lipoglycan includes, per each fucose group, four galactosamine groups, nine glucosamine groups, four galactose groups, one glucose group, and four mannose groups. This lipoglycan can be obtained from a species of the genus Fasciola and/or contain inositol.
The invention further includes a composition including a carrier group coupled to an oligosaccharide isolated from the lipoglycans of the invention. The carrier group can be coupled to the oligosaccharide by a linker (e.g., 2-(4-aminophenyl)ethylamine).
A lipoglycan is a molecule that contains at least one lipid group and at least one carbohydrate group. An isolated lipoglycan is a preparation of a lipoglycan of a particular molecular weight that is at least 60% by weight of the lipoglycan of interest. Of course, the lipoglycan can be isolated and purified to higher levels of purity, e.g., at least 80%, 90%, or 95%, of a composition is the desired lipoglycan. The other 40% can include other macromolecules, such as lipids, proteins, carbohydrates, and lipoglycans not of that particular molecular weight. The lipoglycan can be free of naturally occurring amino acid residues. The molecular weight of a lipoglycan is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.
As used herein, xe2x80x9cprotective immune responsexe2x80x9d means an immune response capable of preventing, reducing, or inhibiting productive infection by a parasite. In the case of a prophylactic composition, the animal or human host has not been infected, thus the composition prevents or inhibits (partially or completely) any productive infection or one or more symptoms of productive infection caused by a subsequent exposure to a parasite. In the case of a therapeutic composition, the animal or human host exhibits an on-going productive infection, and the composition reduces or ends a productive infection. A productive infection is one in which viable parasites can be isolated from a host. A protective immune response includes IgG antibody production and T cell activation. A protective composition, e.g., a vaccine, elicits a protective immune response.
A carrier group is a molecule which, when coupled to an oligosaccharide, helps present the oligosaccharide antigen to a mammalian immune system. Examples of carrier groups include proteins, such as bovine serum albumin (BSA), tetanus toxoid, ovalbumin, and parasite protein.
An adjuvant is a substance that is incorporated into or is administered simultaneously with the compositions of the invention. Adjuvants increase the duration or level of the immune response in an animal after administration of an antigen. An adjuvant can also facilitate delivery of an antigen into the animal or into specific tissues, cells, or locations throughout the body of the animal. Examples of adjuvants include, but are not limited to, incomplete Freund""s, complete Freund""s, and alum; and can contain squalene (e.g., MF59, Chiron Corp, Emeryville, Calif.), monophospholipid A (e.g., DetoxJ, Ribi ImmunoChem Research, Inc., Hamilton, Mont.), saponins (QS-21, Cambridge Biotech, Cambridge, Mass.), non-ionic surfactants (NISV, Proteus, Cheshire, United Kingdom), tocols (U.S. Pat. No. 5,667,784), biodegradable-biocompatible poly(D,L-lactide-co-glycolide) (U.S. Pat. No. 5,417,986), immune-stimulating complexes (ISCOMs), and/or liposomes.
A non-reducing end group, as it pertains to a sugar, means a sugar that does not reduce Benedict""s reagent in the Benedict""s test for reducing sugars; see, e.g., http://www.acp.edu/web/genchem/thedisk/food/bened/bened.htm)
The new isolated lipoglycans are useful in producing therapeutic and prophylactic compositions, such as protective vaccines, against parasites. In turn, the compositions are useful in eliciting a protective immune response against a parasite as detailed in the methods of the invention.
The isolated lipoglycans, compositions, and methods of the invention provide a novel means of preparing and using vaccines against a wide variety of eukaryotic parasites such as flatworms and protozoans. Unlike many parasites in natural infection, the various aspects of the invention offer the ability to stimulate T cell-dependent immune responses in an animal or human host, including parasite-specific IgG production. The methods and compositions of the invention can be used to raise lipoglycan-specific antibodies useful in diagnosis of infection. The isolated lipoglycans of the invention also can be used in diagnostic assays in which the lipoglycans of the invention bind to antibodies present in a biological sample.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The invention provides new therapeutic and prophylactic compositions for use in treating parasitic infections, e.g., by eliciting a protective immune response against parasites. Many parasites are not immunogenic, or not sufficiently immunogenic to produce an effective immune response. The methods of the invention offer a parasite antigen presentation strategy that produces a protective immune response. This strategy includes isolating a lipoglycan from the parasite surface, preparing oligosaccharides from the lipoglycan, coupling the oligosaccharides to a carrier group, and administering the oligosaccharide/carrier group conjugate to the mammal to be vaccinated. These vaccinations offer protective immunity by, at least in part, inducing parasite-specific IgG production, as detailed in the Examples below.
The methods, lipoglycans, and compositions of the invention can be used to vaccinate a mammal against a variety of parasites. One general class of parasites are worms belonging to the phylum platyhelminthes. Platyhelminths include parasitic flatworms of the class trematoda (e.g., worms of the genus Schistosoma, such as S. bovis, S. indicum, S. japonicum, S. mattheei, S. spindale, S. haematobium, S. intercalatum, S. mansoni, or S. mekongi; worms of the genus Fasciola, such as F. hepatica, F. gigantica, and F. jacksoni; and the worm Fascioloides magna) that infect humans and farm animals. Platyhelminths also include tapeworms of the class cestoidea, which infect humans (worms of the genus Taenia) or dogs (worms of the genus Mesocestoides). Another general class of parasites are protozoans such as those of the genus Trichomonas, Tritrichomonas, Leishmania, or Entamoeba. Trichomonas vaginalis is particularly prevalent in the U.S. and can cause symptomatic genitourinary tract infections.
General procedures for isolating the lipoglycans, using the compositions, and performing the methods of the invention, are described below.
Isolation of Lipoglycans from Parasites
Parasites are generally available from vendors, such as the American Type Culture Collection (ATCC), Rockville, Md. For example, T. vaginalis is available as Cat. No. 30001 from ATCC. In addition, F. hepatica matacercariae can be purchased from Baldwin Enterprises, Monmouth, Oreg., or isolated from aquatic snails, a natural host of the parasite. Parasites are also obtainable from various research laboratories, including those supported by the World Health Organization. Alternatively, they can be isolated from natural hosts or the environment (e.g., Fasciola from abattoirs or Fascioloides from deer experiment stations in the field).
The preparation of a parasite at various stages of its life cycles is known in the art. For example, F. hepatica newly excysted juveniles (NEJ) can be isolated according to Hanna, Exp. Parasitol., 50:103-114, 1980. More mature worms can generally be isolated according to Gibbs et al., The Veterinary Clinics of North America: Food Animal Practice, Vol. 2, W.B. Saunders Co., Philadelphia, Pa., pp. 261-275, 1996.
Lipoglycans can be isolated from a parasite by methods known in the art. Typically, the parasites are washed with phosphate-buffered saline.(PBS), and then the low molecular weight lipids on the surface of the parasite, including simple and complex lipids, can be extracted by organic solvents. These crude low molecular weight lipids, which include glycerophospholipids, glycoglycerolipids, and sphingolipids, can be extracted with a solution of chloroform/methanol/water (3:2:1) as described in Turco et al., J. Biol. Chem., 259:3883-3889, 1984; and Bennett, Parasitol., 77:325-332, 1978. Alternatively, the crude lipids can be extracted with a solution of hexane/isopropanol (3:2) as described in Radin, Meth. Enzymol., 72:5-7, 1981.
The choice of extraction method will depend on the parasite. For example, lipoglycan from Fasciola hepatica can be isolated by pre-treatment of parasite tissue using either the chloroform/methanol/water or the hexane/isopropanol extraction method (see Example 1 below) to differentially remove low molecular weight lipids. On the other hand, the lipoglycan from Schistosoma mansoni cannot be isolated using chloroform/methanol/water solvent. However, these low molecular weight lipids can be differentially extracted using the hexane/isopropanol method (see Example 2 below). The extraction with chloroform/methanol/water or hexane/isopropanol in the case of Fasciola hepatica, or hexane/isopropanol in the case of Schistosoma mansoni leaves a residue from which lipoglycan can be extracted with solvent E.
The lipoglycan can be isolated from the low molecular weight lipid residue by extraction with a solution of water, ethanol, diethylether, pyridine, and NH4OH (15:15:5:1:0.017), also called solvent E, as described in Turco et al., supra. The solvent E extract is dried to isolate the lipoglycan. Additional procedures can be performed to further purify the lipoglycan, such as gel filtration, hydrophobic chromatography, and methanol precipitation.
The isolated lipoglycans can be characterized by using SDS-PAGE to determine their molecular weights. In addition, a monosaccharide profile for the carbohydrate portion of the lipoglycan can be obtained by subjecting the lipoglycan to acid hydrolysis and analysis on a high performance anion exchange chromatography system fitted with a pulsed amperometric detector using the manufacturer""s instructions. Such systems and detectors are available from Dionex, Inc., Sunnyvale, Calif.
Producing Oligosaccharide/Carrier Group Conjugates
To produce an antigen useful in a therapeutic or prophylactic composition, such as an anti-parasite vaccine, oligosaccharides are released from the isolated lipoglycan. This can be done using, e.g., standard mild acid hydrolysis or glycosidase treatment. See, e.g., Semprevivo, Carbohy. Res., 177:222-227, 1988. Additional purification (e.g., by column chromatography) of the oligosaccharides can be performed to isolate oligosaccharides of a specific size range (e.g., 800-3000 daltons). These oligosaccharides can include non-reducing end groups, repeating subunits, and/or core portions of the lipoglycan. In addition, the oligosaccharides obtained from a particular LG are expected to contain the same carbohydrate residues as in the LG itself.
The oligosaccharides or mixture of oligosaccharides are then coupled to a carrier group by conventional methods to form effective immunogens because, as haptens, the oligosaccharides alone are likely to be poor immunogens. Carrier groups can be any polypeptide, organic polymer, or smaller molecule that is suitable for administration to a mammal. When coupled to the oligosaccharides, the carrier groups enhance presentation of oligosaccharide epitopes to a mammalian immune system, thereby inducing an immune response specific for the oligosaccharides and, by extension, for the lipoglycan on the surface of the parasite. The use of a mixture of many different oligosaccharides helps to prevent the target eukaryote from adapting and avoiding the immune response.
Any standard chemical linker (e.g., a bi-functional linker containing, for example, reactive amino groups) can be used to couple the oligosaccharides to the carrier group. Examples of such linkers include 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyl hydrazide, and a phenethylamine-isothiocyanate derivative. See, e.g., Lee et al., Vaccine, 14:190-198, 1996; Ragupathi et al., Glycoconjugate J., 15:217-221, 1998; Roy et al., Canad. J. Biochem. Cell Biol., 62:270-275, 1984; and Smith et al., Methods Enzymol., 50:169-171, 1978.
Chemistry and techniques suitable for coupling oligosaccharides to a carrier group such as BSA are known in the art. For example, the carbonyl group of the terminal reducing monosaccharide residue of an oligosaccharide can react with the primary alkylamine group of a linker such as 2-(4-aminophenyl)ethylamine to form an intermediate. This intermediate is then reduced with sodium borohydride to form an unstable intermediate and to facilitate a condensation between the terminal arylamino group of the linker portion of the intermediate and a diazo bridge to residues, e.g., lysine residues, of a polypeptide carrier such as BSA. See, e.g., Zopf et al., Meth. Enzymol., 50:163-169, 1978; and Semprevivo, supra.
While different oligosaccharide molecules derived from the digestion of a single lipoglycan source are coupled to the carrier group using the above methods, oligosaccharides from more than one lipoglycan (e.g., lipoglycans from two species of parasites) also can be linked to a single carrier group. Such multi-specific conjugates are especially useful for the production of broadly protective vaccines.
Preparation of Compositions Containing Oligosaccharide/Carrier Group Conjugates
The compositions can include one or more different types of oligosaccharide/carrier group conjugates. For example, conjugates produced from different lipoglycans can be mixed together in the same composition to produce a cross-protective vaccine composition. In general, the vaccine compositions can be prophylactic (for uninfected individuals) or therapeutic (for individuals already infected).
The compositions optionally include a pharmaceutically acceptable excipient, such as the diluent phosphate buffered saline or bicarbonate (e.g., 0.24 M NaHCO3). The excipients used in the new compositions can be chosen by one of ordinary skill in the art, on the basis of the mode and route of administration, and standard pharmaceutical practice, without undue experimentation. Suitable pharmaceutical excipients and diluents, as well as pharmaceutical necessities for their use, are described, e.g., in Remington""s Pharmaceutical Sciences. An adjuvant, e.g., a cholera toxin, Escherichia coli heat-labile enterotoxin (LT), liposome, or immune-stimulating complex (ISCOM), can also be included in the vaccine compositions.
To formulate the therapeutic compositions, the oligosaccharide/carrier group conjugates can be further purified by standard methods to remove contaminants such as endotoxins, if present. The final conjugate preparation can be lyophilized and resuspended in sterile, deionized water. Appropriate pharmaceutical excipients can then be added.
The therapeutic compositions can be formulated as a solution, suspension, suppository, tablet, granules, powder, capsules, ointment, or cream. In the preparation of these compositions, at least one pharmaceutical excipient can be included. Examples of pharmaceutical excipients include solvent (e.g., water or physiological saline), solubilizing agent (e.g., ethanol, polysorbates, or Cremophor EL7), agent for achieving isotonicity, preservative, antioxidizing agent, lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, light silicic acid anhydride, calcium carbonate, binder (e.g., starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxy methyl cellulose, or gum arabic), lubricant (e.g., magnesium stearate, talc, or hardened oils), or stabilizer (e.g., lactose, mannitol, maltose, polysorbates, macrogols, or polyoxyethylene hardened castor oils). If desired, glycerin, dimethylacetamide, 70% sodium lactate, surfactant, or basic substance such as sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, or trisaminomethane can be added. Biodegradable polymers such as poly-D,L-lactide-co-glycolide or polyglycolide can be used as a bulk matrix if slow release of the composition is desired (see e.g., U.S. Pat. Nos. 5,417,986, 4,675,381, and 4,450,150). Pharmaceutical preparations such as solutions, tablets, granules or capsules can be formed with these components. If the composition is administered orally, flavorings and/or colors can be added.
Administration of Compositions Containing Oligosaccharide/Carrier Group Conjugates
The new compositions can be administered via any appropriate route, e.g., intravenously, intraarterially, topically, by injection, intraperitoneally, intrapleurally, orally, subcutaneously, intramuscularly, sublingually, nasally, by inhalation, intraepidermally, or rectally.
Dosages administered in practicing the invention will depend on factors including the specific vaccine antigen and its concentration in the composition, whether an adjuvant is co-administered with the antigen, the type of adjuvant co-administered, the mode and frequency of administration, and the desired effect (e.g., protection from infection or treatment of an existing infection). Suitable dosages as can be determined by one skilled in the art without undue experimentation. In general, the new compositions can be administered in amounts ranging between 0.01 xcexcg and 1 mg of the conjugate per kilogram body weight. If adjuvants are administered with the compositions, amounts of only 1% of the dosages given immediately above can be used. The dosage range for veterinary use can be adjusted according to body weight.
Administration is repeated as necessary, as determined by one skilled in the art. For example, in prophylaxis a priming dose can be followed by three booster doses at weekly intervals. A booster shot can be given at 8 to 12 weeks after the first immunization, and a second booster can be given at 16 to 20 weeks, using the same formulation. Sera or T-cells can be taken from the individual for testing the immune response elicited by the composition against the parasite (or parasite surface antigens) in vitro. Methods of assaying antibodies, cytotoxic T-cells, or other mediators of immune function against a specific antigen and assaying their ability to kill parasites in vitro are well known in the art, including the ones described in the Examples below. See also, e.g., U.S. Pat. No. 4,656,033; WO 90/02563; Vieira et al., Comp. Biochem. Physiol., 100B:507-516, 1991; Butterworth et al., Immunol. Res., 61:5-39, 1982; Bickle et al., J. Immunol., 128:2101-2106, 1982; and Simpson et al., Infect. Immun., 41:591-597, 1983. Additional boosters can be given as needed. By varying the amount of the immunogen or composition, the immunization protocol can be optimized for eliciting a maximal immune response.
Before administering the above compositions in humans, toxicity and efficacy testing can be conducted in animals. In an example of efficacy testing, mice can be vaccinated via an oral or parenteral route with a composition containing a oligosaccharide/carrier group conjugate antigen. After the initial vaccination or after optional booster vaccinations, the mice (and corresponding control mice receiving mock vaccinations) are challenged with a LD95 dose of the parasite. Protective immunity is then determined by an absence or reduction (e.g., a 70%, 80%, 90%, 95%, 99%, or 100% reduction) in the number of viable parasites. Alternatively, the challenge is a lethal dose, and protective immunity is determined by an absence of lethality. In general, a lethal Fasciola dose in mice is five or more cysts, and a lethal schistosome dose in mice is about 50 or more cercariae.
For example, oligosaccharides from a schistosome lipoglycan can be conjugated to BSA, diluted in PBS, and delivered into mice. As a control, a non-specific oligosaccharide (e.g., maltotriose) can be conjugated to BSA, diluted in PBS, and delivered into mice on the same day. A booster vaccination is give about one month after the first vaccination. About two weeks later, the mice are challenged with 50 to 500 Schistosoma mansoni cercariae. The mice are sacrificed and necropsied about a week after challenge, and the number of viable lung-stage worms in each mouse counted. Protective immunity is conferred by the absence or reduction in number of viable worms in the test mice compared to the presence of viable worms in the control mice. Additional details regarding schistosome infection animal models can be found in Sher et al., J. Inf. Dis. 130:626-633, 1974; and Bergquist et al., Parasitol. Today 14:99-104, 1998.
A vaccine based on a Fasciola lipoglycan oligosaccharide/carrier group conjugate can be tested in like manner as for the schistosome vaccine described above, except that the timing of various steps are adjusted as necessary, and the liver, not the lungs, of sacrificed mice are examined for signs of infection. Again, protective immunity is conferred by the absence or reduction in total number of viable worms in the test mice compared to the presence of viable worms in the control mice, or as a reduction in a symptom associated with infection. Additional details regarding Fasciola infection animal models can be found in Morrison et al., Vaccine 14:1603-1612. 1996; Hughes et al., Res. Vet. Sci. 30:93-98, 1981; and Rajasekariah et al., Exp. Parasitol. 44:233-238, 1978.
Details regarding protozoan infection animal models can be found in Corbeil, Parasitol. Today 10:103-106, 1994; Ghadirian et al., Parasite Immunol. 7:479-487, 1985; Farrell, Exp. Parasitol. 40:89-94, 1976; and Gorczynski, Cell. Immunol. 94:1-10, 1985.
The dose of the conjugate administered to a subject will depend generally upon the severity of the condition (if any), age, weight, sex, and general health of the subject.
Physicians, pharmacologists, and other skilled artisans are able to determine the most therapeutically effective treatment regimen, which will vary from patient to patient. The potency of a specific composition and its duration of action can require administration on an infrequent basis, including administration in an implant made from a polymer that allows slow release of the conjugate. Skilled artisans are also aware that the treatment regimen must be commensurate with issues of safety and possible toxic effect produced by the conjugate or other components in the compositions, such as adjuvants.
Variations
The portions of the lipoglycan molecule which induce protective antibodies can be determined by raising monoclonal antibodies specific for specific regions of the lipoglycan and determining which of these portions of the lipoglycan participate in parasite destruction or elimination. In general, antibodies can be raised by injecting into an animal the immunogenic compositions described herein. Monoclonal antibodies and hybridomas producing them can be cloned and screened (using the original antigen complex as the capture moiety) from a B cell population isolated from the immunized animals using standard methods in the art of molecular biology.
Once antibodies are selected using these screens, the specific oligosaccharide structures to which they bind can be identified by at least two methods. In the first method, the antibodies are used to screen a library of oligosaccharide molecules, each member of the library having a known chemical structure. In the second method, the antibodies are used to xe2x80x9cfish outxe2x80x9d the specific oligosaccharides from a complex mixture of oligosaccharides produced by digesting a lipoglycan using the methods described herein. The structure of the specific oligosaccharides are then identified by chromatographic, spectrometric, or other physical and/or chemical methods known in the art of carbohydrate chemistry.
Another variation involves the selection of carrier groups. Infection with a parasite typically induces specific humoral and cellular immune responses in a host. However, parasite proteins by themselves seldom induce significant resistance to homologous challenge. These parasite proteins therefore can provide ideal carriers to which lipoglycan oligosaccharides can be conjugated. Those proteins which elicit an immune response early in infection can function as efficacious carrier groups for inducing resistance to infection.
The means of conjugating oligosaccharides to carrier groups can also be improved. Several procedures and linkers are available with which to facilitate conjugation of carbohydrates to carrier molecules, as described above.